Glycated proteins assay

ABSTRACT

A solution phase assay of glycated proteins, such as glycated haemoglobin and plasma-soluble glycated proteins, is carried out by reacting the assay sample with a photoluminescent or chemiluminescent marker compound containing a boronic acid group which reacts selectively with the glycated protein. The luminescence of the marker compound is excited at a wavelength at which it is preferentially quenched by the binding of the marked compound to the glycated protein. The quenching in luminescence gives a measure of the degree of glycation of the glycated proteins.

This invention relates to a method for carrying out an assay forglycated proteins, such as glycated haemoglobin.

The level of glycated haemoglobin in blood is a quantity which is usedroutinely to assess diabetic patients. High levels of circulatingglucose lead to high percentages of glycated proteins. For example, in anon-diabetic person, the percentage of haemoglobin molecules that havebeen glycated (i.e. to which glucose has bound non-enzymatically) is4.5-8.0 percent. In a diabetic patient, the levels are much higher(unless treatment is given) and can in severe cases be as much as 20percent. The level of glycated haemoglobin can therefore be used tomonitor treatment.

Virtually all existing methods for the determination of glycatedhaemoglobin in blood samples depend upon the separation of glycatedhaemoglobin using for example chromatography, electrophoresis, or solidphase reagents with washing step, prior to measurement.

For example, Wilson et al. (Clin Chem 39/10, 2090-2097 (1993)) describean assay method for glycated haemoglobin in which the glycatedhaemoglobin is labelled with a soluble polyanionic affinity reagent, andthe anionic complex is then captured with a cationic solid-phase matrix.In this method, the amount of glycated haemoglobin bound to the solidphase matrix is then determined by measuring the quenching by thehaemoglobin of the static fluorescence from an added fluorophore.

EP-A-455225 (Nacalai Tesque, Inc.) discloses a method for determiningthe percentage of glycation of a particular protein. First, the proteinis separated from the sample which contains it by preferentially bindingit to an antibody which is fixed to a solid support. The support is thenwashed with labelled boronic acid, which binds to the glycated site onthe protein. The liquid and solid phases are separated, and the amountof boronic acid bound to the protein in the solid phase can be measured,thereby allowing the number of glycated protein molecules to becalculated.

U.S. Pat. No. 4,861,728 (Becton, Dickinson & Co) describes a method ofdetermining the percentage of glycosylated haemoglobin in blood. In thismethod, the total haemoglobin is separated from the liquid phase bybinding it to a dipstick, and then the glycosylated haemoglobin isreacted with a dihydroxyboryl reagent conjugated to a fluorescent dye.The absorption of incident light by the dye provides a measure of theamount of glycated haemoglobin. This can be compared with the totalamount of haemoglobin, which can be calculated by measuring theabsorption of incident light at the absorption wavelength forhaemoglobin.

A similar method is described in WO 90/13818 (Axis Research AS). In thismethod, the glycosylated haemoglobin-containing sample may be haemolysedto liberate any cell bound haemoglobin. Signal-forming moleculesconjugated to dihydroxyboryl residues are then reacted preferentiallywith the glycosylated haemoglobin, the total haemoglobin is separatedfrom the sample, and the amount of glycosylated haemoglobin is measuredby measuring the amount of signal-forming molecules.

WO 93/18407 (Abbot Laboratories) is concerned with a method of measuringthe amount of glycated haemoglobin in a sample, by reacting the totalhaemoglobin with a fluorescent marker, and measuring the fluorescentquenching due to the total haemoglobin. The glycated haemoglobin is thenseparated from the sample by standard methods (ion capture or solidphase separation), and the fluorescent quenching due to it is measured.The two quenching measurements give a percentage of the totalhaemoglobin which is glycated. The specific binding agent for glycatedhaemoglobin which is employed is coupled to a latex particle or topolyacrylic acid, in order to achieve the separation of glycated andnon-glycated haemoglobin which is essential to the operation of themethod.

Separation assays of this kind are time consuming and thus expensive tocarry out in practice.

A non-separation assay for glycated albumin is described by YukikoHayashi et al. (Clinica Chimica Acta, 149 (1985) 13-19).

The method relies on a specific reaction between the particularfluorophore employed (dansylated phenyl boronic acid) and glycatedalbumin, which results in the enhancement of the fluorescence of thedansylated phenyl boronic acid. The reason for the enhancement is notexplained by the authors, but it clear from the paper that thephenomenon is albumin-specific, i.e., it relies on the specificinteraction between albumin and the particular fluorophore employed.

Albumin in blood has a relatively short lifetime, and therefore it isnot particularly suitable as an marker for levels of glycation.

Also, because of the nature of the particular fluorophore employed(dansylated phenyl boronic acid), excitation of the fluorophore toproduce fluorescence must take place in a region of the spectrum inwhich proteins also absorb strongly (around 350 nm). This results in adisadvantageously high background signal against which fluorescenceenhancement must be measured.

No liquid phase (i.e., non-separation) assay is available for glycatedproteins other than albumin.

We have now found that it is possible to carry out a solution phaseassay of glycated proteins other than albumin, such as glycatedhaemoglobin and plasma-soluble glycated proteins, by reacting an assaysample containing the said glycated protein with a photoluminescent orchemiluminescent marker compound (such as a fluorescent compoundcontaining a fluorescein residue), containing a boronic acid groupcapable of binding with the cis-diol group of the glycated protein,which marker compound is not albumin-specific. The luminescence(typically, fluorescence) of the said residue is then detected at awavelength such that luminescence is preferentially quenched by thebound glycated protein (e.g., glycated haemoglobin), to an extent whichdepends upon on the amount of glycated protein which is present (morespecifically, on the degree of glycation of the protein present). Theamount of the protein present in the solution can thus be determined bythe change in luminescence (e.g. fluorescence) caused by such quenching.

Accordingly, in the first aspect of the invention, there is provided amethod of carrying out an assay for a glycated protein in a sample,which method comprises

carrying out a reaction in solution between an assay sample andphotoluminescent or chemiluminescent marker compound containing aboronic acid group capable of selective binding with the cis-diol groupof a glycated protein, which marker compound is not albumin-specific,

exciting the luminescence in the marker compound, and detecting theresulting luminescence,

wherein the nature of the marker and the nature of the excitation aresuch that the said luminescence occurs at a wavelength at which it ispreferentially quenched by the binding of the said marker compound tothe said glycated protein, and

determining the quenching in luminescence due to the binding of the saidmarker compound to the glycated protein.

The term "photoluminescent" as used herein is intended to include bothphosphorescence and fluorescence, although it is preferred that thephotoluminescent compound is a fluorescent compound. It is preferredthat the fluorophore has a principal excitation wavelength of from 450to 800 nm, (ie. somewhat distant from the principal excitationwavelength of proteins). It is further preferred that the principalfluorescence wavelength is from 450 to 600 nm.

It is particularly preferred that the marker compound contains theresidue of a fluorescent compound such as fluorescein or a fluoresceinderivative, for example carboxyfluorescein or a chlorofluoreacein. Inthis case, the excitation wavelength is preferably approximately 480 nm,and the fluorescence is preferably detected at approximately 520 nm.

Other complex organic molecules which are chemiluminescent orphosphorescent rather than fluorescent can also be used as luminescentmarkers in the method of the invention, provided that theirchemiluminescence or phosphorescence can be selectively quenched bycovalent bonding to a glycated protein such as glycated haemoglobin.

Other suitable fluorophores are the following (the figures shown inparentheses are the principal excitation and fluorescence wavelengths)

naphthofluorescein (λex 600 nm λem 672 nm)

eosin (λex 522 nm λem 543 nm)

erythrosin (λex 528 nm λem 553 nm)

coumarin and umbelliferone (λex 360 nm λem 460 nm) derivatives

rhodamine derivatives

e.g. Rhodamine B (λex 550 nm λem 585 nm)

tetramethyl rhodamine (λex 540 nm λem 570 nm)

texas red derivatives (λex 589 nm λem 615 nm)

lucifer yellow derivatives (λex 420 nm λem 535 nm)

Various BODIPY (4₁ 4-difluoro-4-bora-3a₁ 4a diaza-s-indacine)derivatives

NBD-halide (4-halogeno-7-nitrobenzo-2-oxa-1₁ 3-diazole) derivatives

Lanthanide chelate derivatives

Transition metal chelate derivative, e.g. Ru tris phenanthroline or Rutris bipyridyl derivatives

Phycobiliprotein derivatives

The group capable of selective binding with a cis-diol is a boronic acidgroup. Boronic acids are known to form covalent (but not particularlystable) bonds with the cis-diol groups of glycated proteins. The bondsformed are, however, sufficiently stable to enable the assaydetermination to be made in solution.

Haemoglobin is a known quencher of fluorescence. If a fluorescentmolecule is bound covalently and specifically to glycated haemoglobin,but not to non-glycated haemoglobin molecules, it is possible to derive,without separation, a measure of the level of haemoglobin glycationdirectly from the measured amount of fluorescence quenching. High levelsof glycated haemoglobin will quench the fluorescent signal more than lowlevels. The same effect is observed with other glycated proteins, forexample, plasma-soluble or serum-soluble glycated proteins.

The principal application of the assay method of the invention is in themeasurement of glycation levels of blood proteins linked to the controlof diabetes.

The preparation of boronic acid derivatives of fluorescein is describedfor example, in DE-A-3720736. The derivative employed in the presentinvention is preferably a compound of the formula F--A--B (OH)₂, whereinF is a fluorescein residue, and A is a suitable linking group to linkthe fluorescein residue to the boronic acid group. In a preferredembodiment, the linking group A may be a group of the formula--NH.CS.NH.Ph--, wherein Ph is a phenyl group. In a particular preferredembodiment, the compound has the formula: ##STR1##

A number of preferred embodiments of the invention are described in thefollowing Examples.

The following buffers were employed in the Examples:

Carbonate Buffer

4.2 ml of sodium hydrogen carbonate was dissolved in 500 ml distilledwater and the pH adjusted to 9.0 by addition of solid sodium carbonate.

Assay buffer

7.51 g glycine and 10.16 g magnesium chloride hexahydride were dissolvedin 1000 ml distilled water. The pH of the solution was adjusted to 8.5by addition of 1.0M sodium hydroxide solution.

Sample lysing buffer

2 ml of a lysing detergent (TM TRITON X-100) was dissolved in 100 ml ofassay buffer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of ΔF versus % HbA1 (Corning test kit) and depictsresults of Example 2.

FIG. 2 is a graph of ΔF/OD versus % HBA1 (Corning test kit) and depictsresults of Example 2.

FIG. 3 is a graph of ΔF versus % HbA1 (Corning test kit) and depictsresults of Example 3.

FIG. 4 is a graph of ΔF versus % HbA1 (HPLC) and depicts results ofExample 4.

FIG. 5 is a graph of ΔF versus % HbA1 (HPLC) and depicts results ofExample 5.

EXAMPLE 1

A fluorescein-boronic acid compound of formula I above was prepared bythe following method. Fluorescein isothiocyanate (10 mg),m-aminophenylboronic acid (4 mg) and triethylamine (3 mg) were mixed in0.9 ml methanol, and 0.1 ml distilled water. The mixture was stirred forone hour at room temperature and then a further 4 mg ofm-aminophenyl-boronic acid was added. Stirring was continued for afurther hour, and the solution was then purified by thin layerchromatography (eluent dichloromethane:methanol, 9:1).

The green band (Rf 0.2-0.3) was eluted from the silica with methanol,and the resultant solution stored at -20° C. 0.5 ml of the methanolsolution was diluted in 4.5 ml carbonate buffer and the optical densityrecorded at 492 nm. It was established that the optical density wasproportional to the concentration, the optical density being 8.78×10⁴×the concentration in mols/liter.

The solution was diluted in the assay buffer to a concentration of 5nmols/l.

EXAMPLE 2

Glycated Haemoglobin Assay

Whole blood samples were collected from patients and stored at 4° C.with an EDTA anti-coagulant.

The samples were centrifuged at 3000 rpm for 5 minutes. 50 μl aliquotswere added to 450 μl sample lysing buffer and vortex mixed.

20 μl aliquots of the lysate were added to 2 ml samples of thefluorescent tracer solution prepared in Example 1, vortex mixed, andleft in the dark for 30 minutes at room temperature. The samples werethen read on a fluorimeter (Perkin Elmer LS20™ equipped with a flowcell), at 520 nm (excitation frequency 480 nm). Similar runs were madewithout sample addition as controls and the total quenching for each ofthe sample runs was determined by difference.

Total haemoglobin content of each of the measured samples was alsoquantified by UV spectrophotometry using a CECIL 300™ UV instrument at405 nm, and the ratio calculated of the level of fluorescence quenchingto the total haemoglobin content (as measured by ΔF/OD, where ΔF is thedecrease in signal measured at 520 nm caused by quenching, and OD is theoptical density measured at 405 nm).

The glycated haemoglobin level (% HbA1) of the same blood samples wasmeasured using a commercially available test kit (Corning), and theresults are tabulated in Table I. FIGS. 1 and 2 respectively showgraphically the correlation between ΔF, and % HbA1, and between ΔF/OD,and % HbA1. The clear correlation in FIG. 2 demonstrates that the singlephase fluorescence quenching method of the present invention may be usedas an accurate measure of glycated haemoglobin level of a blood sample.The correlation in FIG. 1 illustrates that ΔF alone may in somecircumstances (namely, when the total haemoglobin level is known not todiffer widely between samples) be a sufficiently accurate measure ofHbAl content.

EXAMPLE 3

Plasma Assay

Whole blood samples were collected from patients as in Example 2 andstored at 4° C. with an EDTA anti-coagulant.

The samples were centrifuged at 3000 rpm for 5 minutes. 50 μl aliquotsof the supernatant plasma was diluted to 500 μl with 450 the same bufferas used in Example 2, and vortex mixed.

20 μl aliquots of the mixed solution were added to 2 ml samples ofsolution prepared as in Example 1, vortex mixed, and left in the darkfor 30 minutes at room temperature. The samples were then read as inExample 2.

The glycated haemoglobin level of the original blood samples was againmeasured using the Corning test kit. The results are tabulated in Table2. The same results are shown graphically in FIG. 3.

In this case, fluorescence quenching is not caused directly by glycatedhaemoglobin, since the level of glycated haemoglobin in the plasmasample is negligible. The quenching is clearly caused by the presence inthe plasma of some other glycated protein. Table 2 and FIG. 3 clearlyillustrate that a strong correlation also exists between the glycatedhaemoglobin level in the original blood samples and fluorescencequenching caused by the presence of this material in the supernatantplasma. For clinical purposes, it is not necessary to know the precisenature of glycated plasma protein which causes the fluorescencequenching, provided that the amount of fluorescence quenching correlateswith the level of glycated haemoglobin level in the original bloodsamples.

EXAMPLE 4

Whole blood

Whole blood samples were collected from patients and stored at 4° C.with an EDTA anti-coagulant.

The samples were rotated for 10 minutes. 50 μl aliquots were added to450 μl sample lysing buffer and vortex mixed. These were left at roomtemperature for 1 hr.

20 μl aliquots of the lysate were added to 2 ml of the fluorescenttracer solution as prepared in Example 1, vortex mixed, and left in thedark for 30 minutes at room temperature. The samples were then read on afluorimeter (Perkin Elmer LS20™ equipped with a flow cell), at 520 nm(excitation frequency 480 nm). Similar runs were made without sampleaddition as controls and the total quenching for each of the sample runswas determined by difference.

The glycated haemoglobin level (% HbA1c) of the same blood samples wasmeasured using a commercially available Table 3. FIG. 4 showsgraphically the correlation between ΔF and % HbA1c. The correlationshown in FIG. 4 demonstrates that the single phase fluorescencequenching method of the present invention may be used as an accuratemeasure of glycation level of a blood sample.

EXAMPLE 5

Filter paper blood spot

30 μl samples were collected from patients, spotted onto absorbentfilter paper and stored at room temperature.

Filter paper blood spots, cut using a hole-punch, were added to 2 ml ofthe fluorescent tracer solution as prepared in Example 1, vortex mixedand left in the dark for 60 minutes at room temperature. The sampleswere then vortex mixed and read on a fluorimeter (Perkin Elmer LS20™equipped with a flow cell), at 520 nm (excitation frequency 480 nm).Similar runs were made without sample addition as controls and the totalquenching for each of the sample runs was determined by difference.

The glycated haemoglobin level (% HbA1c) of the same blood samples wasmeasured using a commercially available test kit (HPLC method) and theresults are tabulated in Table 4. FIG. 5 shows graphically thecorrelation between ΔF and % HbA1c. The correlation in FIG. 5demonstrates that the single phase fluorescence quenching method of thepresent invention may be used as an accurate measure of glycation levelof a blood spot sample.

EXAMPLE 6

Use of other fluorophores

An umbelliferone-boronic acid compound was prepared by the followingmethod.

7-hydroxy-4-methylcoumarin-3-acetic acid succinimidyl ester (10 mg) indimethylformamide (100 μl), and m-aminophenylboronic acid (5 mg) in pH9.0 carbonate buffer (100 μl) were mixed and stirred for 1 hour in thedark. The solution was purified by thin layer-chromatography (eluentchloroform:methanol, 4:1).

A band (Rf 0.79-0.82) was eluted from the silica with methanol. Analiquot of the methanol solution was diluted tenfold in carbonate bufferpH 9.0. The concentration of product was calculated using the opticaldensity at 360 nm and an extinction coefficient of 17,000.

The quenching of the prepared umbelliferone-boronic acid derivative wasmeasured using the procedure given in Example 3, but with 100 nMconcentration of the umbelliferone-boronic acid derivative and 200 μlaliquots of samples diluted tenfold. The results are shown in Table 5 .

It will of course be understood that the invention may be put intopractice in many other ways in addition to those specifically outlinedabove. In particular, the nature of the fluorophore may be varied,appropriate changes being made to the excitation frequency.

                  TABLE 1    ______________________________________    Sample          Meas. (F.).sup.3                    Δ F.                             Hb OD  Ratio  Corning.sup.1    ______________________________________    1     724.50    94.30    0.380  248.16 4.80    1     728.20    90.60    0.380  238.42 4.80    2     719.30    99.50    0.359  277.16 5.50    2     721.40    97.40    0.359  271.31 5.50    3     727.80    91.00    0.380  239.47 7.60    3     726.80    92.00    0.380  242.11 7.60    4     711.70    107.10   0.396  270.45 8.90    4     715.20    103.60   0.396  261.62 8.90    5     696.50    122.30   0.378  323.54 11.20    5     698.70    120.10   0.378  317.72 11.20    6.sup.2          701.60    117.20   0.376  311.70 11.60    6.sup.2          700.50    118.30   0.376  314.63 11.60    7     691.60    127.20   0.382  332.98 12.20    7     691.30    127.50   0.382  333.77 12.20    8     682.00    136.80   0.403  339.45 15.10    8     683.20    135.60   0.403  336.48 15.10    9     658.70    160.10   0.391  409.46 16.70    9     661.20    157.60   0.391  403.07 16.70    ______________________________________     .sup.1 results of Corning test are given as % HbA1     .sup.2 Sickle cell sample     .sup.3 Total fluorescence (no sample) value = 818.80

                  TABLE 2    ______________________________________    Sample   Meas. (F.)    Δ F.                                   Corning    ______________________________________    1        700.2         95.1    5.3    1        697.6         97.7    5.3    2        705.4         89.9    5.7    2        697.6         97.7    5.7    3        673.4         121.9   6.0    3        679.6         115.7   6.0    4        669.1         126.2   6.6    4        670.6         124.7   6.6    5        638.0         157.3   7.6    5        639.0         156.3   7.6    6        656.4         138.9   7.9    6        660.2         135.1   7.9    7        657.3         138.0   8.1    7        667.5         127.8   8.1    8        646.7         148.6   8.3    8        638.0         157.3   8.3    9        638.0         157.3   8.4    9        645.9         149.4   8.4    10       609.7         185.6   8.8    10       604.5         190.8   8.8    11       608.5         186.8   9.1    11       605.3         190.0   9.1    12       619.7         175.6   9.4    12       618.1         177.2   9.4    13       638.6         156.7   9.6    13       643.0         152.3   9.6    14       613.9         181.4   10.5    14       622.0         173.3   10.5    15       620.7         174.6   10.7    15       629.3         166.0   10.7    16       543.3         252.0   11.4    16       551.1         244.2   11.4    17       588.4         206.9   11.5    17       593.7         201.6   11.5    18       585.1         210.2   12.3    18       587.2         208.1   12.3    19       602.4         192.9   13.3    19       603.6         191.7   13.3    20       572.8         222.5   13.6    20       571.3.        224.0.  13.6.    ______________________________________     Total fluorescence (no sample) value = 795.30

                  TABLE 3    ______________________________________    SAMPLE  % HbA1cc   FLUORESCENCE INDEX                                        Δ F.    ______________________________________    1       5.1        592.8            157.2    2       6.2        598.8            151.2    3       7.1        567.2            182.8    4       8.0        575.1            174.9    5       8.6        566.6            183.4    6       9.0        569.6            180.4    7       10.1       572.7            177.3    8       10.8       557.9            192.1    9       11.2       548.0            202.0    10      11.7       554.4            195.6    ______________________________________     TOTAL FLUORESCENCE = 750

                  TABLE 4    ______________________________________    SAMPLE  % HbA1cc   FLUORESCENCE INDEX                                        Δ F.    ______________________________________    1       5.1        560               90    2       6.2        548              102    3       7.1        514              136    4       8.0        525              125    5       8.6        496              154    6       9.0        502              148    7       10.1       518              132    8       10.8       499              151    9       11.7       510              140    ______________________________________     TOTAL FLUORESCENCE = 650

                  TABLE 5    ______________________________________                           FLUORESCENCE                           QUENCHING INDEX    SAMPLE    (approx) % HbA1c                           (Δ F.)    ______________________________________    High      13           61    Medium    7            53    Low       <1           42    ______________________________________

We claim:
 1. A method of carrying out an assay for a glycated protein ina sample, which method comprises:carrying out a reaction in solutionbetween an assay sample and photoluminescent or chemiluminescent markercompound containing a boronic acid group capable of selective bindingwith the cis-diol group of a glycated protein, but not exclusively withalbumin, and, without separation of the reaction product from thesolution, exciting the luminescence in the marker compound, anddetecting the resulting luminescence, wherein the nature of the markerand the nature of the excitation are such that the said luminescenceoccurs at a wavelength at which it is preferentially quenched by thebinding of the said marker compound to the said glycated protein, anddetermining the quenching in luminescence due to the binding of the saidmarker compound to the glycated protein.
 2. A method as claimed in claim1, wherein the glycated protein is glycated haemoglobin, or aplasma-soluble glycated protein.
 3. A method as claimed in claim 1 orclaim 2, wherein the sample is a blood sample, a plasma sample or aserum sample.
 4. A method as claimed in claim 1, wherein thephotoluminescent compound is a fluorescent compound.
 5. A method asclaimed in claim 4, wherein the fluorescent compound has a principalexcitation wavelength of at least 450 nm.
 6. A method as claimed inclaim 5, wherein the fluorescent compound has a principal excitationwavelength of from 450 to 800 nm.
 7. A method as claimed in any one ofclaims 4 to 6, wherein the fluorescent compound has a principalluminescence wavelength of from 450 to 600 nm.
 8. A method as claimed inclaim 7, wherein the said principal luminescence wavelength isapproximately 520 nm.
 9. A method as claimed in claim 4, wherein thefluorescent compound contains a fluorescein residue.
 10. A method asclaimed in claim 9, wherein the fluorescent compound is a group of theformula F--NH--CS--NH--Ph--B(OH)₂, wherein Ph is a phenyl group and F isa fluorescein residue.
 11. A method as claimed in claim 9, wherein thefluorescent compound is a compound of the formula: ##STR2##
 12. A methodas claimed in claim 1, wherein the glycated protein is glycatedhaemoglobin.